Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.9 (glucose-6-phosphatase)
3,081 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth-walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.
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PMID:Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions. 16 28

Since infections with Schistosoma mansoni cause marked histopathological changes in the liver of the host, the effect of this infection on the hepatic drug-metabolizing function was investigated. Severity of Schistosomiasis was determined by worm counts, duration of infection, egg counts and liver weight increases. To overcome difficulties in homogenizing the livers of infected animals, preincubation of the squashed tissues with collagenase and hyaluronidase was used to prepare homogenates. Key component enzyme activities of the hepatic microsomal drug-metabolizing enzyme system (NADPH-cytochrome c reductase and cytochrome P-450) as well as the representative drug-metabolism activities (aminopyrine N-demethylase, aniline hydroxylase, and benzpyrene hydroxylase) were measured for the whole liver and found to be markedly reduced. However, the measurement of microsomal marker enzyme activities (cytochrome b5 and glucose-6-phosphatase) showed significant elevation. To obtain more precise information about the effect of the schistosome infection on the hepatic drug-metabolizing enzyme system, the total activities of microsomal drug-metabolizing enzymes were related to the total microsomal marker enzyme activities in the homogenate.
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PMID:Effect of Schistosoma mansoni infection on the hepatic drug-metabolizing capacity of mice. 18 61

Periportal (pp) or perivenous (pv) liver parenchymal cells from female adult Uje: WIST rats were isolated after retro- or antegrade digitonin infusion followed by collagenase perfusion in the opposite direction. The morphological results revealed a distinct acinar-related destruction of the pv- or pp-zone by digitonin. The remaining cells of the respective other zone showed a good structural maintenance. After subsequent conventional collagenase perfusion the yield, viability and structural integrity of the isolated hepatocytes were high. The zonal cell separation was indicated by significant differences in the pp marker glucose-6-phosphatase and the pv marker glutamine synthetase found in the isolated pp or pv cell populations. Under our experimental conditions including the use of female rats, the alanine aminotransferase and glutamate dehydrogenase as well as ethylmorphine N-demethylase and ethoxycoumarin O-deethylase activities were evenly distributed in both preparations. Under stimulating conditions the capacity for urea synthesis was similar in both pv and pp cells.
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PMID:Biochemical and morphological studies on perivenous and periportal liver parenchymal cells from female rats isolated by digitonin-collagenase method. 168 Jul 46

In the presence of phenobarbital (PB) at 3 mM, hepatocytes isolated from adult rats by a collagenase-perfusion technique survived well on plastic dishes for at least 49 days after initiation of primary culture. PB at concentrations less than 3 mM was ineffective for the maintenance of hepatocytes, and the maintenance of them was attained only in the continuous presence of 3 mM PB. The hepatocytes surviving in the presence of 3 mM PB were morphologically indistinguishable from the hepatocytes after 1-day attachment period, except for the presence of prominent nucleoli in the former. Although both the albumin secretion and tyrosine aminotransferase (TAT) activities of the cells decreased gradually up to day 7 with time in culture, both were thereafter maintained at relatively high levels at least up to day 35 of primary culture. The addition of 10 microM dexamethasone caused a 3-5-fold induction in TAT activity, and the cells were capable of responding to the hormone in this manner at least up to day 28 of primary culture. Furthermore, the cells also had glucose-6-phosphatase activity, even though the level of this enzyme activity was relatively low as compared with that of TAT activity. Survival of hepatocytes in the presence of 3 mM PB was further enhanced by simultaneous addition of dexamethasone (10 microM) and insulin (10 micrograms/ml). The sensitivity of hepatocytes to 3'-methyl-4-dimethylaminoazobenzene (0.24 mM) was remarkably reduced by treatment with PB at 3 mM. PB treatment decreased efficiently the falling rate of total cytochrome P-450 content, but did not induce P-450PB, which is the specific form of cytochrome P-450 induced by PB, in primary cultured hepatocytes. On the other hand, 3-methylcholanthrene (MC, 10 microM) caused an increase of both contents of total cytochrome P-450 and P-450MC, which is the specific form of cytochrome P-450 induced by MC, in primary cultured hepatocytes. However, MC was ineffective for the maintenance of hepatocytes in primary culture. The possible biological actions of PB on primary cultured hepatocytes are discussed on the basis of the experimental data obtained.
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PMID:Long-term survival of functional hepatocytes from adult rat in the presence of phenobarbital in primary culture. 286 57

Cells from kidney proximal tubules have been successfully isolated, characterized, and cultured from male Fischer 344 rats between 150-400 g using a two-step collagenase perfusion. The cells undergo high levels of DNA synthesis and mitosis in both serum free media (with an without hormone supplementation) and media containing 10% fetal bovine serum. Confluent monolayers were observed between 5 to 7 days after seeding 2 X 10(5) cell/35 mm collagen-coated plate. Approximately 50% of the total kidney and 70% of the cortex was isolated using this technique. The viability of the isolated tubules was 75 +/- 8% and the estimated number of viable cells was 12 +/- 3 X 10(6) cells. At the time of isolation greater than 90% of the isolated tubules and cells were positive for gamma glutamyltransferase (GGT), periodic acid-schiff (PAS), and glucose-6-phosphatase (G-6-Pase). Both GGT and G-6-Pase decreased rapidly during the first 3 days in primary culture as assessed by histochemistry. Ultrastructurally the isolates consisted of cells with numerous microvilli and mitochondria. The size and number of microvilli decrease rapidly in primary culture. The morphologic and biochemical evidence suggests that the primary isolates and cultures are proximal tubular in origin.
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PMID:Kidney proximal tubular cells isolated by collagenase perfusion grow in defined media in the absence of growth factors. 288 90

Suspensions of freshly isolated hepatocytes were prepared by collagenase perfusion of livers of adult Fischer 344 female rats. The cells were injected into the dorsal fascia of 2/3 partially hepatectomized syngeneic hosts (10(6) cells per injection site) and were monitored from 3 days to 3 months after injection. Brown nodules developed at the transplantation site. Histologic examination of the nodules revealed that the hepatocytes were arranged in cords and clusters surrounded by fibrovascular connective tissue. Bile ductules were also seen. Hepatocytes were positive for glucose-6-phosphatase. Staining for gamma-glutamyltranspeptidase showed that the parenchymal hepatocytes were mostly (approximately 95%) negative, whereas bile ductules were positive. These histochemical findings were seen in hepatocytes up to 3 months after transplantation and did not vary with the age of the transplants. Electron-microscopic examination of the transplanted nodules demonstrated that the cells maintained the characteristics of hepatocellular cytoplasmic structure. The relationship between the bile canaliculi and the stromal vessels was found to be similar to the bile canaliculi and hepatic sinusoid polarity seen in the normal liver. Autoradiographic analysis showed that a fraction of the transplanted cells was active in DNA synthesis. This system may become a tool in the study of survival and neoplastic transformation of hepatocytes as a result of exposure to X-irradiation and chemical carcinogens.
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PMID:Morphologic and histochemical analysis of hepatocytes transplanted into syngeneic hosts. 610 19

Primary cultures of liver cells isolated from adult rats by trypsin and collagenase perfusion techniques were carried out to compare cytologic and biochemical properties between the differently prepared cells. Trypsin-dispersed cells consisted of comparatively smaller cells, whereas collagenase-dispersed cells consisted of larger cells. The cell attachment efficiency on culture day 1 was about twice as high in the liver cells prepared with collagenase than those prepared with trypsin. Mature hepatocytes isolated by collagenase perfusion could be maintained in the primary culture for a longer period than those isolated by trypsin perfusion. Epithelial-like clear cells started to grow much earlier in the primary culture of the trypsin-dispersed liver cells than in that of the collagenase-dispersed liver cells. Earlier proliferation of epithelial-like clear cells could not be induced by in vitro trypsinization of the collagenase-dispersed liver cells. Both kinds of enzymatically prepared liver cells showed albumin production and exhibited glucose 6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9, G6Pase) and tyrosine aminotransferase (L-tyrosine: 2-oxoglutarate amino-transferase, EC 2.6.1.5, TAT) activities for 1 week in the primary culture. Albumin production was higher in the liver cells prepared with collagenase than those prepared with trypsin, whereas G6Pase activity was almost the same between them. TAT activity up to culture day 2 was about 3-fold higher in the liver cells prepared with collagenase than in those prepared with trypsin. Combined supplementation of dexamethasone (1 X 10(-5)M) and insulin (10 micrograms/ml) consistently improved the cell attachment efficiency and was very effective in the maintenance of mature hepatocytes in both types. Furthermore, these hormones enhanced the albumin production and TAT activity in both types.
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PMID:Comparison of cytologic and biochemical properties between liver cells isolated from adult rats by trypsin perfusion and those isolated by collagenase perfusion. 614 85

Putative preneoplastic hepatocytes were isolated from male Fischer 344 rats treated with a single dose of diethylnitrosamine, 2-acetylaminofluorene feeding, and partial hepatectomy (Solt-Farber model). The isolation procedure involved, after collagenase dispersion of the liver, separation of the hepatocytes into small- and large-cell fractions by centrifugal elutriation, and subsequent selection of cells deficient in asialoglycoprotein receptor(s) by plating onto asialofetuin (ASF)-coated plates. The number of cell surface binding sites for the asialoglycoprotein receptor was measured with both asialoorosomucoid and ASF as ligands. There was a 50% reduction of binding sites for both ligands in the original cell suspensions obtained from preneoplastic livers. The reduction in receptor binding sites was most pronounced in the large cell fraction (less than or equal to 30% of control value) after separating the original cell suspension by elutriation into small and large cell fractions. Immunohistochemical studies showed a lack of asialoglycoprotein receptor in preneoplastic (i.e., hyperplastic foci) areas. These areas were entirely super-imposable with glucose-6-phosphatase-deficient areas and partially overlapped the gamma-glutamyltranspeptidase-positive areas in serial liver sections. The attachment of preneoplastic hepatocytes to ASF-coated tissue culture dishes was greatly impaired, and the number of gamma-glutamyltranspeptidase-positive cells on the ASF dishes was reduced to less than 7% as compared to 45 to 70% on the collagen-coated plates. Thus, the lack of asialoglycoprotein (asialofetuin) surface receptors and the increased size of the early preneoplastic hepatocytes are characteristics that can be used to separate the preneoplastic cell population from normal liver cells.
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PMID:Isolation of preneoplastic rat liver cells by centrifugal elutriation and binding to asialofetuin. 620

The cytochemical localization of glucose-6-phosphatase (G6Pase) and its biochemical quantification were studied in isolated and cultured adult rat parenchymal cells. Appropriate technical conditions were chosen to assume adequate ultrastructural preservation and retention of enzyme activity. Isolated hepatocytes separated by collagenase perfusion were shortly fixed in glutaraldehyde and entrapped in a pellet of fibrin. Frozen sections, 50 microns in thickness were incubated for cytochemical demonstration of G6Pase, in a slightly modified Wachstein-Meisel medium. Hepatocytes in culture, fixed for 1 min in glutaraldehyde, were impregnated in a 10% cryoprotective glycerol solution and quickly frozen in liquid nitrogen at -170 degrees C in order to induce penetration of the substrate. In these conditions, a homogeneous distribution of the enzyme was observed in both isolated and cultured cells. The cytochemical reaction appears continuous in the smooth and rough endoplasmic cisternae and in the nuclear envelope. Lead phosphate deposits, although evenly distributed, are reduced in intensity after 48 h culture. Biochemical determinations reveal the presence of a high specific enzymatic activity in isolated cells (108 nmolP/min/mg proteins), which decreases in culture, respectively to 70 and 50% of the original value, after 24 and 48 h culture. G6Pase induction by glucagon was obtained after 48 and 72 h in culture.
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PMID:Glucose-6-phosphatase distribution in isolated and cultured adult rat hepatocytes. 626 35

Although the activity of glucose-6-phosphatase in rat liver is altered markedly following the administration of a variety of hormones in vivo, it is not certain whether the hormones act directly on the hepatocyte. To study this problem hepatocytes were isolated by a collagenase-perfusion technique and cultured on collagen gel/nylon mesh membranes. The activity of glucose 6-phosphatase in cells cultured with fetal calf serum and with Dulbecco's modified Eagle's medium or Leibovitz L-15 medium decreased to less than 10-30% of the activity in freshly isolated cells by 96 h. However, when L-15 plus newborn or fetal calf serum was supplemented with glucagon (10(-6)M), epinephrine (10(-6)M), triiodothyronine (10(-6)M), and dexamethasone (10(-5)M) (L-15-GETD), the activity of glucose-6-phosphatase was maintained so that, after 144 h, the activity was at least 80% of that detected in freshly isolated cells. In cells cultured in L-15 plus serum for 72 or 96 h and then in L-15-GETD, glucose-6-phosphatase increased 30-50% over that in control cultures after 24 h. Insulin, which decreases glucose-6-phosphatase activity when administered to intact animals, also decreased the glucose-6-phosphatase activity in cultured hepatocytes to 20-50% of that in controls.
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PMID:Effects of hormones on the activity of glucose-6-phosphatase in primary cultures of rat hepatocytes. 631 98


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